Spontaneous Ca2+ release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload-induced Ca2+ release (SOICR) can result in Ca2+ waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here, we show that a point mutation E4872A in the helix bundle crossing (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, RyR2 Ca2+ activation. Introducing metal-binding histidines at this site converts RyR2 into a luminal Ni2+ gated channel. Mouse hearts harboring an RyR2 mutation at this site (E4872Q+/−) are resistant to store overload-induced Ca2+ waves and completely protected against Ca2+-triggered VTs. These data show that the RyR2 gate directly senses store Ca2+, explaining RyR2 store Ca2+ regulation, Ca2+ wave initiation, and Ca2+-triggered arrhythmias. This novel store-sensing gate structure is conserved in all RyRs and inositol 1,4,5-trisphosphate receptors.

The 12.6-kDa FK506-binding protein (FKBP12.6) is considered to be a key regulator of the cardiac ryanodine receptor (RyR2), but its precise role in RyR2 function is complex and controversial. In the present study we investigated the impact of FKBP12.6 removal on the properties of the RyR2 channel and the propensity for release or store overload-induced Ca 2؉ release (SOICR). FK506 increased the amplitude and decreased the frequency of SOICR in HEK293 cells expressing RyR2 with or without FKBP12.6, indicating that the action of FK506 on SOICR is independent of FKBP12.6. As with recombinant RyR2, the conductance and ligand-gating properties of single RyR2 channels from FKBP12.6-null mice were indistinguishable from those of single wild type channels. Moreover, FKBP12.6-null mice did not exhibit enhanced susceptibility to stress-induced ventricular arrhythmias, in contrast to previous reports. Collectively, our results demonstrate that the loss of FKBP12.6 has no significant effect on the conduction and activation of RyR2 or the propensity for spontaneous Ca 2؉ release and stress-induced ventricular arrhythmias.

A number of RyR2 (cardiac ryanodine receptor) mutations linked to ventricular arrhythmia and sudden death are located within the last C-terminal approximately 500 amino acid residues, which is believed to constitute the ion-conducting pore and gating domain of the channel. We have previously shown that mutations located near the C-terminal end of the predicted TM (transmembrane) segment 10, the inner pore helix, can either increase or decrease the propensity for SOICR (store-overload-induced Ca2+ release), also known as spontaneous Ca2+ release. In the present study, we have characterized an RyR2 mutation, V4653F, located in the loop between the predicted TM 6 and TM 7a, using an ER (endoplasmic reticulum)-targeted Ca2+-indicator protein (D1ER). We directly demonstrated that SOICR occurs at a reduced luminal Ca2+ threshold in HEK-293 cells (human embryonic kidney cells) expressing the V4653F mutant as compared with cells expressing the RyR2 wild-type. Single-channel analyses revealed that the V4653F mutation increased the sensitivity of RyR2 to activation by luminal Ca2+. In contrast with previous reports, the V4653 mutation did not alter FKBP12.6 (FK506-binding protein 12.6 kDa; F506 is an immunosuppressant macrolide)-RyR2 interaction. Luminal Ca2+ measurements also showed that the mutations R176Q/T2504M, S2246L and Q4201R, located in different regions of the channel, reduced the threshold for SOICR, whereas the A4860G mutation, located within the inner pore helix, increased the SOICR threshold. We conclude that the cytosolic loop between TM 6 and TM 7a plays an important role in determining the SOICR threshold and that the alteration of the threshold for SOICR is a common mechanism for RyR2-associated ventricular arrhythmia.